Semi-field evaluation of a volatile transfluthrin-based intervention reveals efficacy as a spatial repellent and evidence of other modes of action

Presently, the most common malaria control tools–i.e., long lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS)–are limited to targeting indoor biting and resting behaviors of Anopheles mosquito species. Few interventions are targeted towards malaria control in areas where transmission is driven or persists due to outdoor biting behaviors. This study investigated a volatile pyrethroid-based spatial repellent (VPSR) designed to bridge this gap and provide protection from mosquito bites in outdoor spaces. Southern Province, Zambia, is one such environment where outdoor biting is suspected to contribute to malaria transmission, where people are active in the evening in open-walled outdoor kitchens. This study assessed the VPSR in replica kitchens within a controlled semi-field environment. Endpoints included effects on mosquito host seeking, immediate and delayed mortality, deterrence, blood feeding inhibition, and fertility. Host-seeking was reduced by approximately 40% over the course of nightly releases in chambers containing VPSR devices. Mosquito behavior was not uniform throughout the night, and the modeled effect of the intervention was considerably higher when hourly catch rates were considered. These two observations highlight a limitation of this overnight semi-field design and consideration of mosquito circadian rhythms is recommended for future semi-field studies. Additionally, deterrence and immediate mortality were both observed in treatment chambers, with evidence of delayed mortality and a dose related response. These results demonstrate a primarily personal protective mode of action with possible positive and negative community effects. Further investigation into this primary mode of action will be conducted through a field trial of the same product in nearby communities.

were fully informed of the risks and voluntarily provided informed oral consent. Their employment was not contingent on participation in the study. They did not receive additional compensation or incentives for the study but were paid at their normal pay rates for their work. Vector control measures have a large impact on malaria burden, accounting for an estimated 81% of total 50 malaria reduction between 2000-2015 (1). The existing interventions, long lasting insecticide-treated nets (LLINs) and 51 indoor residual spraying (IRS), counter indoor late-night biting and indoor resting behaviors. These bionomic traits are 52 generally common in many key Anopheles vectors of malaria including An. gambiae s.s. and An. funestus, the primary 53 vectors in much of sub-Saharan Africa. However, susceptible biting patterns have been observed to shift in response to 54 interventions (2-4). In addition, molecular identification has revealed unexpectedly high Anopheles species diversity in 3 many settings demonstrating greater complexity in transmission dynamics (5-7). These shifts in species compositions, 56 densities, and bionomic traits are not well documented and may be highly spatially heterogenous, with one model 57 estimating that Africans in general are experiencing 10% fewer of their mosquito bites while in bed or indoors in 2018 58 compared to 2003 (8). In many settings, this shift in biting patterns has resulted in increased mosquito exposure 59 outdoors or earlier in the evening (4,(9)(10)(11)(12)(13)(14). For vector control efforts, these behavioral shifts and other gaps in 60 protection are identifiable in many settings and are becoming more relevant to malaria transmission in areas where 61 much of the malaria burden has been reduced such as Zambia's Southern Province where this study was conducted. 62

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Community protection is a vital aspect of existing interventions including LLINs and IRS; by reducing local 63 mosquito populations and therefore exposure of the community as a whole, the insecticidal action of these 64 interventions can provide significant protection to a user's unprotected neighbors (15). This community effect 65 somewhat depends on the resistance status of local mosquitoes, and is a vital aspect of the continued effectiveness of 66 these interventions (16)(17)(18)(19). While LLINs and IRS provide significant community effects through control of local malaria 67 vectors, they both specifically target indoor biting and resting behaviors. Additional tools that target other mosquito 68 behaviors may be required to provide personal and community protection in additional settings. There are no official 69 recommendations for interventions to be deployed in these spaces, such as outdoors and in the early evening, where 70 people may be at risk of mosquito biting (20). Structural improvements, such as closing eaves and screening windows 71 and doorways of homes, are possible alternatives to traditional indoor interventions but are impractical or inapplicable 72 for many peridomestic and outdoor spaces (21). Larval source management allows public health officials to reduce local 73 mosquito populations, but is impractical over large areas and doesn't directly target outdoor biting (20,22). 74 Outdoor transmission is relevant in many low malaria settings, due in part to human behaviors in the early 75 evening and morning which can expose them to mosquito activity (4,23). Outdoor human-mosquito interactions are 76 quite variable, highlighting the advantages of flexibility, portability, and ease of use in interventions that target these 77 behaviors. Volatile pyrethroid-based spatial repellent (VPSR) interventions incorporate these advantages and have 78 shown promise in reducing mosquito landing behavior in prior studies (24)(25)(26), in addition to increasing mosquito 79 mortality (27)(28)(29), and some evidence of reducing blood feeding behavior (27). The Personal Insect Repellent Kit (PIRK) is 80 a volatile pyrethroid spatial repellent (VPSR) developed by Widder Bros., Inc. The pyrethroid active ingredient 81 transfluthrin has been shown to cause mortality/knockdown effects in addition to repellency in Anopheles mosquitoes 82 (25,30,31). These outcomes are of epidemiological importance, with the undesirable possibility of repelled mosquitoes 83 diverting to nearby hosts (32), and knockdown or kill effects possibly reducing transmission in a mechanism similar to 84 the community effect provided by LLINs and IRS (33). 85 This study used a semi-field system at Macha Research Trust, southern Zambia, to evaluate the PIRK with 86 entomological outcomes in mind. The system is in a setting with seasonal and very low malaria transmission, with 87 outdoor mosquito biting behavior and high ITN coverage. Much of the outdoor biting is thought to occur in the evenings 88 and mornings while families are gathered in open-walled kitchen huts. The PIRK devices could therefore reduce 89 mosquito interactions in these spaces. The study design utilized the semi-field enclosure to conduct controlled release 90 and recapture experiments to measure endpoints beyond mosquito landing. Study endpoints were designed to measure 91 landing rate, repellency, and knock down during active PIRK use, in addition to delayed effects on blood feeding, 92 mortality, and fecundity after exposure. These study endpoints were employed to assess the efficacy and longevity of 93 PIRK devices. 94

96
This study was approved by the institutional review board at the University of Notre Dame (Protocol #: 18-05-97 4675) and by the local IRB at Macha Research Trust (IRB #: IRB0007649). Study participants were Macha Research Trust 98 entomology staff who were fully informed of the risks and voluntarily provided informed oral consent. Their 99 employment was not contingent on participation in the study. They did not receive additional compensation or 100 incentives for the study but were paid at their normal pay rates for their work. 101

102
The Personal Insect Repellent Kit (PIRK) is a volatile pyrethroid spatial repellent (VPSR) developed by Widder 103 Bros., Inc. (Fig 1). Each device consists of two 25x25cm sheets and serves as a passive emanator of the volatile 104 5 pyrethroid transfluthrin to provide an area of protection from mosquito activity for up to a month or longer without 105 replacement. The devices were deployed within the semi-field kitchen structures by hanging them under two opposite 106 eaves of the structure, 1.5 meters from the floor (Fig 1). Two devices were deployed to each kitchen (one per side). 107

108
The semi-field system is a large enclosure walled with a fine mesh to prevent the ingress of local insects or the 109 egress of test mosquitoes whilst allowing temperatures to largely equilibrate with the external environment (Fig 1). The 110 two test chambers are separated by an unused third test chamber, and each chamber is isolated from the other by 111 interior walls made of the same fine mesh material. Each chamber measures approximately 10m by 10m, with a lower 112 ceiling of fine mesh about 3m above the ground. The entire enclosure sits under a simple sealed plastic roof for 113 protection from the elements. Each chamber is surrounded by a narrow ditch filled with water and a mild surfactant, 114 which prevents crawling insects that might prey on mosquitoes from reaching the interior of the chambers. During the 115 experiments, the cement floors of the chambers were covered in white cloth which was wetted with water before each 116 experiment. These cloths served to increase the relative humidity within chambers while providing a backdrop to easily 117 find dead mosquitoes. This design allowed for the simultaneous evaluation of PIRK against a baseline control using 118 mosquitoes reared from the same generation in an insectary. 119 The test huts located in each chamber represent shelters used in the area. For this study, the upper half of test 123 shelter walls were removed to replicate the design of local kitchen shelters (Fig 1). These shelters have 2m x 2.5m floors, 124 with brick walls roughly 1m tall except in the doorway, an additional 1m of open sides, and a grass roof. 125

Study design
126 This experiment used a simple 2x2 Latin square rotational design between the test chamber (PIRK) and control 127 chamber (negative control) to account for chamber and weekday effects. The human collectors stayed in the same 128 chamber on each night to enable the collector and chamber effects to be coupled as a single source of bias. Experiments 129 were conducted every third or fourth night (on Monday and Thursday nights) to provide a wash out period between 130 replicates and allow the transfluthrin and any host associated odors to dissipate between rotations. 131 Experiments were conducted from December 2019 through April 2020. Temperature and humidity were 132 recorded at 5-minute intervals for the duration of experimental nights using a data logger (Onset HOBO). External 133 rainfall and moon phase were recorded categorically for each night. Experiments took place over 32 nights, including 134 two nights of baseline collection with no PIRKs in place. Ten nights were dedicated to testing ten separate fresh sets of 135 PIRK devices. Four of these sets were tested weekly for five weeks over the remaining twenty nights. Between 136 timepoints, these devices were kept freely hanging out of direct sunlight above an open office window. 137 cages and experimental groups were kept in 30cm x 30cm BugDorm cages. Reared mosquitoes were provided with 10% 141 sucrose solution ad libitum. These cages were held in the insectary at 27 degrees C and 80% humidity until transfer to 142 the experimental chambers. Approximately 250-300 2-5-day old female mosquitoes were selected from these colonies 143 for release into per experimental chamber on a given night. Sucrose was removed from cages to starve experimental 144 mosquitoes four hours prior to release. 145

Cage setup
146 Mosquitoes were selected in the early afternoon and placed into separate cages for each chamber before 147 experiments commenced. PIRK devices were deployed to the appropriate chamber before 17:00, while the opposite 148 chamber served as a no-device control. At 17:00, the chambers were prepared for the nightly replicate. This included 149 filling the perimeter troughs with water and a mild surfactant and laying out and wetting white cloth on the floors of 150 each chamber. At 18:00, mosquitoes were moved to the experimental chambers from the insectary and released from 151 their cages, signaling the start of an experimental replicate (Fig 2). 152 offered to host-seeking mosquitoes at 1-day post-exposure, 24 hours after mosquitoes were released into experimental 154 chambers. Egg-laying rates were measured overnight after 4-days post-exposure, with hatch rates measured the 155 following morning. Delayed mortality was noted for all days starting at 1-day post-exposure for deterred and host-156 seeking mosquitoes. 157 Host-seeking behavior 161 Mosquitoes were recaptured from within the shelters by trained entomologists performing human landing 162 collection (HLC). This is the gold standard for mosquito collection, where collectors use a mouth aspirator to collect 163 mosquitoes that land on them (34). Collections took place overnight, with mosquitoes collected and counted separately 164 by hour from 18:00-06:00. Collectors were provided coffee and listened to music or radio to aid in staying awake. All 165 mosquitoes caught by HLC were moved to the insectary at the end of each hour for additional experiments. Host seeking 166 behavior measured by HLC constituted the primary experimental endpoint and was calculated per night by dividing the 167 total HLC recapture in a chamber by the number of mosquitoes released. Hourly host seeking behavior was calculated by 168 dividing the HLC recapture for a single hour by the number of mosquitoes remaining in the chamber at the start of that 169 hour. 170

171
Following HLC collections, additional collectors entered each chamber at 08:00 and collected all remaining alive 172 and dead mosquitoes. Two collectors per chamber actively searched for remaining mosquitoes using mouth aspirators, 173 with one searching inside the hut and the other the remaining portions of the chamber including the perimeter ditch. 174 Collectors rotated between chambers halfway through at 09:00, finishing clearing chambers at 10:00. The locations of 175 these mosquitoes were noted as inside/outside the shelter, with survivors moved to the insectary and knocked down 176 mosquitoes counted and discarded. Deterrence was informed by the proportion of released mosquitoes found alive 177 outside of the hut in these morning collections. Knocked down or dead mosquitoes were not monitored for recovery, 178 and direct mosquito mortality was calculated as the proportion of released mosquitoes found knocked down or dead. 179 Overall recovery rate 180 The overall recovery rate from chambers was included as a general measure of experimental bias and to detect 181 effects which were not captured by study endpoints. The overall recovery rate was calculated by dividing the sum of 182 mosquitoes recovered from the chamber (host-seeking, deterred, and knocked down) by the number of mosquitoes 183 released into the chamber the previous night. On a small number of experimental nights (n = 5/64), this recovery rate 184 was slightly higher than 100%, possibly due to miscounting or mosquito survival inside the chambers between replicates. 185 For these chambers, the number of released mosquitoes was amended to yield a recovery rate of 100%. 186

187
In the insectary, HLC-captured and deterred mosquitoes were separately followed for five days post-exposure 188 (d.p.e.) to measure delayed mortality effects of PIRK exposure. Additionally, host seeking (HLC captured) mosquitoes 189 were offered a bloodmeal from an anesthetized mouse at roughly 18:00, or 12-24-hours post-capture to measure 190 inhibition of blood feeding behavior (disarming). These mosquitoes were sugar-starved for four hours prior to the 9 bloodmeal. The numbers of bloodfed females were counted and provided wetted filter papers for egg laying three days 192 later (Fig 2). The numbers of laid eggs were counted and deposited into fresh larval pans; the hatched larvae were 193 counted the next day and discarded. These data were used to post-exposure blood feeding, egg laying (fecundity), and 194 egg hatch rates (fertility). 195

Statistical analysis
196 Generalized linear mixed models with an appropriate error distribution and a log link function were used for 197 analysis. Host seeking, knockdown, and deterrence were analyzed using a Poisson distribution, which included an offset 198 term to adjust for the number of mosquitoes exposed to the outcome (e.g., released into each chamber). Fixed effects 199 included treatment and the age (in weeks after opening) of the PIRK device, chamber, temperature, and humidity with 200 the date of experiment included as a random effect to account for day-to-day variation in mosquito behavior and other 201 unmeasured factors contributing to experimental variation. An interaction between treatment and PIRK age was added 202 to measure the effect of time on PIRK efficacy. Model coefficients were exponentiated and reported as rate ratios with 203 the control set as the reference. The remaining endpoints gathered in the insectary (blood feeding, delayed mortality, 204 fecundity) were modeled using a binomial distribution with the addition of cage density as a fixed effect. Models were 205 evaluated and selected based on Akaike information criterion (AIC), with some fixed effects dropped from specific 206 models when their addition reduced model fit. All data analysis was conducted in R version 4.0.2. Data was cleaned, 207 summarized, and plotted using the tidyverse packages 'tidyr', 'dplyr', and 'ggplot2'. Generalized linear models were 208 generated and analyzed with the 'lme4' and 'arm' packages. 209

210
Over the course of data collection from December 2019 through April 2020, the mean temperature during the 211 hours of experiments was 21 degrees C (s.d. = 1.6), decreasing slightly over time. Mean nightly humidity was 83.9% (s.d. 212 = 13.5%), only dipping below 75% for a few nights in December and early 2020. In general, the early period had warmer 213 and sometimes dryer nights (Fig S1). 214

215
A total of 8885 mosquitoes were released across 34 nights (261/night) in untreated chambers, compared to 216 7848 across 30 nights (262/night) in treatment chambers. The additional four nights represent a baseline period which 217 was not included in comparison models. An identical number of mosquitoes were released into both chambers on a 218 given night. The overall recovery rate, defined as the number of mosquitoes that were recovered from the chambers by 219 all experimental endpoints, was similar between treatment (84.9%) and control (89.7%) (RR: 0.95 [0.90 -1.00], p = 220 0.061), as well as between chambers (86.4% vs 88.5%; (RR: 1.02 [0.99 -1.06], p = 0.25). There was no observed 221 significant effect of treatment, chamber/volunteer, or any other predictors on the recovery rate (full model in Table S1). 222 Host-seeking behavior of mosquitoes -protective efficacy 223 In total, 7033/7974 (88.2%) of mosquitoes released in control chambers were captured by HLC, compared to 224 4319/6662 (64.8%) in PIRK chambers. The greatest protective efficacy (PE) was observed using fresh PIRK devices, with 225 PE remaining but generally decreasing through the five tested weeks (Fig 3 and Fig 4).  11 chambers is displayed on the Y axis cumulatively by hour along the X axis. Each panel represents experimental nights 241 with the corresponding PIRK age in weeks, up to five weeks past opening. 242 Nightly temperature and humidity had no effect on control HLC recapture rates, but both were associated with reduced 243 overnight PIRK efficacy (scaled temperature RR: Mosquito knock-down was also elevated in treatment chambers (9.4% of all recovered mosquitoes, n = 257 626/6662)) compared to control chambers (3.3%, n=261/7974). The largest difference was observed using freshly 258 opened PIRKs (15.5% in test chambers vs 2.3% in control), and these effects subsided in the five weeks after opening. 259 Overall, there was no observable chamber effect on knock-down (6.1% vs 6.0% between chambers for all experimental 260 nights). PIRK was associated with a substantial increase in the proportion of mosquitoes knocked down .68], p < 0.001), with the effect decreasing with PIRK age (weekly "PIRK age" RR: 0.71 [0.65 -0.78], p < 0.001) (Table  262   S1). The ratio of deterrence relative to knockdown increased with weekly PIRK age, with fresh PIRK devices resulting in 263 higher knockdown relative to deterrence compared to older devices, which trend towards heightened deterrence 264 overall (Fig 5). 265 Fig 5. Ratio of deterrence compared to mortality associated with PIRK exposure in the semi-field system. The ratio of 266 deterrence (captured alive outdoors and abbreviated to "Det.") between PIRK and control chambers was divided by the 267 ratio of knockdown (KD) between PIRK and control chambers and plotted on the y axis. Results were separated by age 268 category on the x axis. The dotted horizontal line refers to a ratio of 1 (no change between groups). Ratios greater than 269 one indicate higher deterrence relative to mortality. 270

271
The survival of mosquitoes recaptured while host-seeking (HLC captured) and moving away from the PIRK 272 (deterred) was observed separately for five days post-exposure (d.p.e). Overall mortality of control mosquitoes was 273 4.66% (n = 319/6846) at one d.p.e. and 22.5% (n = 1543/6846) at five d.p.e. This was slightly elevated among PIRK 274 exposed mosquitoes at one d.p.e, 6.48% (n = 391/6036) and five d.p.e. 30.7% (n = 1856/6036). 275 Among host-seeking mosquitoes, control mortality was 4.40% at one d.p.e. (n = 273/6201) and 18.6% at five 276 d.p.e. (n = 1155/6201) and was again slightly higher in the PIRK exposed mosquitoes: 6.07% (n = 262/4319) and 24.6% (n 277 = 1061/4319), respectively. Conversely, mortality of deterred mosquitoes in control chambers was 7.13% after one 278 d.p.e. (n = 46/645) followed by 60.2% at five d.p.e. (n = 388/645), compared to 7.51% (n = 129/1717) and 46.3% (n = 279 795/1717) at each time point in PIRK chambers (Fig 6). 280 Fig 6. Risk ratios of secondary outcomes to PIRK exposure. Y axes represent the risk ratio of each outcome for PIRK 281 exposed mosquitoes compared to control chambers. Ratios were calculated based on the age category of the PIRK 282 devices separated on the X axis by color and symbol and denoted in the figure legend. The dotted horizontal line 283 represents risk ratio of 1, or no change between PIRK exposure and control. Numbers greater than one indicate 284 outcomes which are more common in PIRK chambers compared to controls, while numbers lower than one indicate 285 outcomes which were lessened in PIRK chambers. 286 13 PIRK exposure was associated with increased mortality at one day (OR: 2.42 [1.43 -4.09, p = 0.001) and five 287 d.p.e. (OR: 1.50 [1.15 -1.96], p = 0.003) compared to controls. Higher temperature during the night of capture was 288 associated with slightly higher mortality after one day (scaled temperature OR: 1.25 [1.01 -1.55], p = 0.043) and five 289 days (OR: 1.17 [1.02 -1.33], p = 0.025). The deterred population of mosquitoes experienced similar mortality compared 290 to the host-seeking population after 24 hours and increased mortality at five d.p.e. (OR: 4.54 [1.58 -13.02], p = 0.005). 291 The interaction between population and treatment was borderline significant after five days (OR: 0.34 [0.11 -1.01], p = 292 0.053). This interaction term can be interpreted as the effect of PIRK on mortality of deterred mosquitoes relative to the 293 overall effect of PIRK on all mosquito delayed mortality. Caged mosquito density was included as a predictor in all 294 models, with increased density nearing a significant association with decreased mortality at 5 d.  Table S2. 298 Blood feeding behavior of host-seeking mosquitoes 299 Blood feeding rates of host-seeking mosquitoes were measured 12-24 hours post exposure and were slightly 300 lower in PIRK exposed mosquitoes during experimental nights which used freshly opened PIRKs (83.0% compared to 301 95.8% in control mosquitoes; Fig 6). Blood feeding rates were not reduced in PIRK-exposed mosquitoes at other time 302 points (94.8% in PIRK chambers, 92.6% in control), and model outputs indicate that PIRK exposed mosquitoes blood fed 303 at a reduced rate compared to controls (RR: 0.92 [0.87 -0.98], p = 0.013) (Table S2). Models estimate this effect 304 diminishes after PIRK aging (weekly "PIRK age" RR: 1.02 [1.00 -1.05]), and the raw data reveals no impact on blood 305 feeding rates at one week after opening (97.1% feeding rate in mosquitoes from PIRK chambers, 95.3% in control). 306

315
The endpoints collected in this trial were designed to measure the personal protection offered by the Personal 316 Insect Repellent Kit (PIRK) and other possible effects on Anopheles gambiae vectors that could provide community 317 protection if applied at scale. In addition to landing rates measured by HLC, which have been significantly reduced by 318 transfluthrin-based interventions in prior studies (24,35,36), an additional endpoint -deterrence -was measured by 319 morning outdoor capture of living mosquitoes that were not captured throughout the night. These fates of non-host-320 seeking mosquitoes in the presence of PIRK -and similar devices -are relevant in a field setting, with deterred 321 mosquitoes possibly diverted to surrounding unprotected households in a manner that may be dose-dependent 322 (26,32).The results from these semi-field trials of PIRK indicate that PIRK is associated with a reduction of approximately 323 35-40% in overnight mosquito host-seeking behavior in chambers with freshly opened devices, with the effect declining 324 slightly over time but persisting through the testing period lasting five weeks, with additional effects of increased 325 deterrence and mortality compared to unexposed mosquitoes. Host-seeking reduction was observed at all time points, 326 providing evidence for efficacy up to five weeks and suggesting possible efficacy beyond that period. Mosquito mortality 327 was most strongly associated with fresh PIRK devices and mortality trended towards deterrence as the devices aged, 328 possibly related to a dose response as the remaining transfluthrin in the devices declined. The intervention appeared to 329 have little lasting impact on disarming blood feeding behavior, fecundity, or fertility. These results indicate that the 330 intervention functions as expected through the primary mode of action in reducing landing, but the impacts are not 331 limited to reduction in landing and the impact on disease transmission may be considered based on the accumulation of 332 these effects. 333 It is possible that host-seeking behavior is over-estimated in this study, as the closed semi-field design forces 334 non-host-seeking mosquitoes to remain within 10 meters of host-seeking cues from the human landing collectors 335 throughout the night. In control chambers, hourly landing rates were higher in the first hour than all other hours, 336 highlighting an hourly difference in host-seeking avidity that may be an artifact of the semi-field setting, considering that 337 the natural circadian rhythms of An. gambiae s.s. generally peak after midnight. In models which consider the hourly 338 HLC recapture rate, the predicted effect of PIRK is elevated roughly 50% above the observed all-night reduction of host-339 seeking, driven by the large difference in activity in the first hour which then "trails off" throughout the night (Fig 4). 340 Maximum response in the first hour has been observed in other semi field studies (37,38), and some authors have used 341 multiple releases throughout the night to maintain mosquito biting pressure (39). This straightforward change to semi-342 field design should be considered in future designs to investigate these hourly differences specifically to determine if 343 they are more closely related to mosquito behavior within this closed system or product efficacy. With these 344 considerations, the results of this study can be interpreted with the nightly efficacy acting as a more conservative 345 estimate compared to the hourly results. 346 The secondary endpoints measured in this experiment were chosen to reflect outcomes of epidemiological 347 importance in the field and from modeling studies (33). Mosquito mortality or reductions in fitness have been observed 348 in prior studies of transfluthrin (27)(28)(29), providing a mechanism for community protection through overall suppression 349 of mosquito populations and reduced age structures (40,41). Deterrence and knockdown were both elevated in PIRK 350 chambers and may be dose-dependent, with the ratio of mortality to deterrence highest when testing fresh PIRK devices 351 ( Fig 5). Notably, the exposure-related mortality was largely observed in the perimeter ditches of treatment chambers. It 352 is possible these mosquitoes were repelled by the PIRK devices and would have escaped in a natural setting but were 353 prevented from doing so by the confines of the chamber, leading to over-estimation of mortality in this study. Their 354 accumulation in the perimeter ditches also prevented the differentiation between mortality and knockdown effects. 355 The fates of host-seeking and deterred mosquitoes are also relevant in the context of community protection. In 356 addition to the acute mortality/knockdown which may occur during exposure, delayed impacts on mosquito survival can 357 contribute to community protection. In this study, mortality of host-seeking and deterred mosquitoes was observed for 358 five days. A large increase in mortality was observed among "deterred" mosquitoes in both treated and untreated 359 chambers after five days which was not present at one day, likely driven by the lack of a bloodmeal provided to these 360 mosquitoes at the one-day time point. Mortality was significantly increased in PIRK-exposed HLC-captured mosquitoes 361 at both time points, but this deleterious effect of PIRK was very nearly significantly reversed among deterred mosquitoes 362 after five days (OR: 0.34 [0.11 -1.01], p = 0.053). This suggests that deterred mosquitoes could be less negatively 363 impacted by PIRK compared to host-seeking mosquitoes, possibly due to lower exposure of active ingredient outside of 364 the huts. It is also possible that -considering they missed twelve hours of feeding opportunities -the 'deterred' 365 mosquitoes found in control chambers represent a particularly unfit subset of the original population, resulting in 366 abnormally high mortality. This alternative explanation provides further support for a nightly multiple release 367 experimental design. This deterrence effect should be further studied, as deterred mosquitoes appear capable of 368 enduring PIRK exposure and may divert to other nearby hosts. This finding also supports the ability of this semi-field 369 system to estimate repellency and/or deterrence mechanisms, although an idealized design would be substantially 370 larger than the expected area of effect of the tested device. 371 Prior studies of transfluthrin have utilized proxy measurements for blood feeding such as HLC (42), or allowed 372 mosquitoes to freely bite to measure reductions in blood feeding (28). It has been suggested in Aedes mosquitoes that 373 landing and biting inhibition might differ (43), and separating these endpoints allows for host-seeking and probing 374 behaviors to be considered separately. This disarming endpoint measured by a prolonged blood feeding inhibition even 375 after exposure is particularly important to capture in semi-field systems, since biting behavior cannot be well quantified 376 during field trials involving HLC or other trapping methods. In this study, blood feeding rates among host-seeking 377 mosquitoes observed 12-24 hours post exposure were slightly, but significantly depressed in mosquitoes exposed to 378 PIRKs, with the effect observed to entirely diminish by the first week after opening. This reduction in blood feeding 379 behavior appears to be short-lived but should continue to be further studied in the presence or immediate aftermath of 380 PIRK exposure, rather than 12-24 hours post exposure, to measure for how long after exposure mosquitoes are 381 disarmed and if disarmament provides a community effect by delaying feeding cycles (27,33). Following successful 382 feeding, egg-laying and hatch rates were slightly higher among PIRK exposed mosquitoes overall but varied by PIRK age 383 without following a clear trend. Models suggested that both rates were driven by the mosquito density in experimental 384 cages, which was considerably higher among control mosquitoes, rather than PIRK status. 385 Temperature and humidity also appear to play a role in mosquito behavior and PIRK efficacy, with higher nightly 386 temperature and humidity associated with reduced PIRK efficacy in these experiments. In hourly analysis, higher 387 humidity was associated with generally higher host-seeking across both chambers, while higher temperature and 388 lowered humidity were associated with reduced PIRK efficacy. It is unclear why the association of humidity with PIRK 389 efficacy is reversed in hourly and nightly analysis; it's possible that it is a byproduct of improved model fit due to higher 390 data resolution, or an indication of mosquito behavioral patterns. Overall, these experiments were conducted in cooler 391 than optimal temperatures for volatile pyrethroids with a mean nightly temperature around 21C. Increased 392 temperatures resulting in reduced PIRK efficacy contrasts with other studies that have shown improved efficacy at 393 higher temperatures (26). 394

395
The results of this semi-field study suggest that the PIRK device, a passive emanator of the pyrethroid 396 transfluthrin, could be useful as a malaria control tool. Over the course of experimental nights, human landing was 397 reduced at all time points over the five-week observation period after unsealing the devices, with evidence for 398 heightened mortality transitioning towards deterrence effects over the use of the intervention. Landing rates were 399 reduced up to and including five weeks past opening, with further duration of effect unknown from these trials. Overall 400 rates of deterrence and mortality decreased over the five-week period in addition to the trend towards deterrence, 401 suggesting a possible dose response. In general, there was very little impact on disarming, fecundity, or fertility, except 402 in the case of fresh PIRKs where there was a small but significant decrease in blood feeding (a disarming effect). The 403 data gathered in this study also support possible improvements for semi-field experimental design, primarily shorter 404 recapture timeframes and repeated releases during experimental nights to better mimic a field environment where 405 approaching mosquitoes are more consistently naïve to the active ingredient being tested. We also recommend that 406 temperature and humidity is always monitored when evaluating the efficacy of personal protection tools. Measurements were taken from a weather station (Onset HOBO) adjacent to the semi-field enclosure. Values are 531 plotted on the same axis, with humidity reported as relative humidity (percentage) and temperature reported in Celsius. 532 Table S1. Model coefficients for primary experimental outcomes. All models were mixed effect generalized linear 533 models (GLMER) with a Poisson (log) link function. Each model included the log-transformed number of mosquitoes 534 released into the chamber as the exposure term, except for the hourly host-seeking model which uses the log-535 transformed number of mosquitoes remaining in the chamber at each hour. Models were assessed by AIC and 536 coefficients which were dropped to enable model convergence are denoted with a dash '-'. AIC and degrees of freedom 537 for the null model are displayed in parenthesis after the values for each fitted model. Coefficients which are not relevant 538 to a specific model are denoted with an NA. Date of experiment was included in all models as a random effect. P values 539 are coded, with '***' representing p values < 0.001, '**' representing p values between 0.001 and 0.01, '*' between 0.01 540 and 0.05, and '.' representing nearly significant p values between 0.05 and 0.1. & Temperature and Humidity were 541 centered and scaled around their mean values for all models. 542 Table S2. Model coefficients for delayed mortality and blood feeding behavior. All models were mixed effect 543 generalized linear models (GLMER) with a binomial (logit) link function. Models were assessed by AIC and coefficients 544 which were dropped to enable model convergence are denoted with a dash '-'. AIC and degrees of freedom for the null 545 model are displayed in parenthesis after the values for each fitted model. Coefficients which are not relevant to a 546 specific model are denoted with an NA. Date of experiment was included in all models as a random effect. P values are 547 coded, with '***' representing p values < 0.001, '**' representing p values between 0.001 and 0.01, '*' between 0.01 548 and 0.05, and '.' representing nearly significant p values between 0.05 and 0.1. & Coefficients denoted with this symbol 549 were centered and scaled around their mean values prior to model fitting. % The age of treatment was considered as a 550 numeric predictor in all models except the blood feeding model, where a binary factor (fresh vs not fresh) was used 551 instead to better model the observed behavior. 552